Udobre et al. Afr. J. Pharmacol. Ther. 2013.

2(3): 83-87

African Journal of Pharmacology and Therapeutics Vol. 2 No. 3 Pages 83-87, 2013
Open Access to full text available at http://www.uonbi.ac.ke/journals/kesobap/

Research Article

Effect of Methanol Leaf Extract of Nauclea latifolia

on Albino Mice Infected with Plasmodium berghei
berghei
Aniefiok S. Udobre a,*, John A. Udobang b, Anwanabasi E. Udoh b, Victor U. Anah a,
Akaninyene E. Akpan a, and Goodnews E Charles a
a Department
b Department

of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo, Nigeria

of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Nigeria

_____________
* Corresponding author: Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of
Uyo, P.M.B. 1017 Uyo, Nigeria; Tel: +234-802-7672367; Email: aniefiokudobre@yahoo.com
Background: In Nigeria the leaf decoction of Nauclea latifolia is taken to treat malaria and sexually transmitted
diseases. This study intends to generate a scientific data in support of the traditional use of the leaves in malaria
treatment.
Objective: To investigate the antiplasmodial effect of the methanol extract of the leaves of Nauclea latifolia on
chloroquine sensitive Plasmodium berghei berghei in experimentally infected albino mice.
Materials and Methods: The fresh leaves of Nauclea latifolia were collected, dried under shade, ground into powder
and macerated in methanol for 72 hrs. The dried extract was stored at -4 C for use. Thirty (30) mice were divided
into five groups (A,B,C,D,E). Group A received 10 ml/kg/day of distilled water (negative control). Groups B, C and D
received 370, 740 and 1110 mg/kg/day of the extract respectively. Group E received 1.2 mg/kg/day of artesunate
(positive control). This experiment was repeated for suppressive, prophylactic and curative tests.
Results: The extract produced considerable antiplasmodial activity in all the three tests evaluated compared to the
standard drug (artesunate). The extract reduced parasitaemia significantly (p<0.05) in a dose dependent manner.
Bioactive constituents of the plant could be responsible for the antiplasmodial activity
Conclusion: The result of the study supports the need for continued search for components of traditional medicine as
potential antimalarial agents.
Keywords: Malaria, Nauclea latifolia, mice, Plasmodium berghei berghei
Received: April, 2013
Published: October, 2013
1. Introduction
There are about 380,000 species of plants in our world
of which 260,000 belong to the higher plants; only 10%
of the higher plants have been studied to a significant
extent (Sandberg, 1976).
Nauclea latifolia is a multi-stemmed tree of up to 12 m
tall. It has an open canopy. Flowers are joined by their
calyxes. The fruit is a syncarp.

A KeSoBAP Publication 2013. All rights reserved.

The young leaf contains mild alkaloids with analgesic

property. The leaf preparations are taken as a
vermifuge and diuretic, for amenorrhoea, antimalarial
and sterility. It is hypotensive and hypoglycemic. In
Corte de Voire, it is taken for jaundice and blood in
urine, intestinal troubles and incipient hernia . In Ghana,
it is taken for fever and jaundice. In Democratic
Republic of Congo, leaf preparations are used externally
on oedemas. The Igala of Northern Nigeria use a boiling
water infusion of the leaf on swellings and skin rashes.
The Tenda of South East Senegal crush and cook the

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leaves to make plasters for wound dressings. The leaves
are deemed to have haemostatic property and are put
on circumcision wounds and on haemorrhoids and
hernias. The leaf extract is taken with chewed alligator
pepper to relief malaria dysentery and diarrhoea. The
leaf decoction is taken to treat sexually transmitted
diseases (Burkill, 1997).
In a study, the aqueous extracts of Nauclea latifolia
leaves (0.25-2.0 mg/ml) paralyzed T. columbrifomis
larva in a concentration-dependent manner (Asuzu and
Njoku, 1996). Ethanol stem extracts of Nauclea latifolia
decreased the level of parasitaemia in a dose-dependent
manner in mice experimentally infected with
Trypanosome brucei (Madubunyi, 1995). In another
study Nauclea latifolia extract was effective against
nociception, inflammation and pyrexia in rats and mice
thus partially justifying its use traditionally in the
treatment of uncomplicated malaria and painful
condition (Abbah et al, 2010).
Malaria is a mosquito-borne infectious disease of
humans and other animals caused by protozoa of the
genus Plasmodium (WHO, 2011). At present, 40% of the
world population lives in malaria endemic regions
(Snow, 2005). In 2001 about 700,000 lives of children
below the age of five were lost to malaria in Africa. The
disease is responsible for many medical complications
like low birth weights in infants and increase in sudden
abortion and stillbirth in pregnant women (Joda et al,
2005). Malaria is responsible for about 66% of all clinic
visits in Nigeria. It causes the death of an estimated
250,000 children under the age of five every year.
According to UNICEF findings, about 60% of deaths
especially those of children in the Specialist Hospital in
Bauchi, Nigeria are caused by malaria (UNICEF, 2009).
There is increase in the resistance of P. falciparum to
antimalarial drugs. This development has compromised
the efficacy of the available antimalarial drugs such as
chloroquine, Fansidar and even artemisinin. Records
have shown that there are no drugs that can offer
protection against malaria in all the regions of the world
(WHO, 2011).

2.2 Extraction and Phytochemical Analysis

1.0 kg of the powdered leaves was macerated with
methanol for 72 hr at room temperature (27+2 C) with
agitation 3 times daily to enhance the extraction
process. The extracts were filtered out using filter
paper (Whatman No. 1). The filtrates were evaporated
to dryness using rotary evaporator and water bath at 40
C. The concentrated extracts were then transferred to
air-tight containers, corked and preserved in the
refrigerator until required.
The
methanol
extracts
were
screened
for
phytochemicals using the standard methods of analysis
by Trease and Evans (2009) and Sofowora (2008).
2.3 Breeding of Animals
Ninety (90) male albino mice weighing between 14-20 g
were collected from the Animal House, Department of
Pharmacology, University of Uyo, Nigeria. The mice
were bred, food and water provided ad libitum. The
animal house was cleaned and disinfected regularly and
the sawdust changed three times a week.
2.4 Parasite Inoculation
A chloroquine sensitive strain of P. berghei berghei
(ANKA) was obtained from the National Institute of
Medical Research (NIMER), Lagos and was maintained
by sub passage in mice (Basir et al, 2012).
Each mouse used in the experiment was inoculated
intraperitoneally with 0.3 ml of infected blood
containing about 1x107 Plasmodium berghei parasitized
erythrocytes. The inoculum consisted of 5x107
Plasmodium berghei parasitized erythrocytes per ml.
This was prepared by determining both the percentage
parasitaemia and the erythrocytes count of the donor
mouse and diluting the blood with isotonic (normal)
saline in proportions indicated by both determinations:
red blood counts and percentage parasitaemia (Odetola,
1980).
2.5 Evaluation of Antiplasmodial Activity

The research on Nauclea latifolia reported herein

therefore is a contribution to the research efforts
towards identifying new potential treatments for
malaria. Previous studies on Nauclea latifolia led to the
development of a tablet dosage form from the water
extract of this plant. The tablet produced had good
mechanical properties like hardness but poor friability
and disintegration (Emeje et al, 2005).
2. Materials and Methods
2.1 Collection of Plant Materials
The fresh leaves of Nauclea latifola were collected in
September, 2011 from Ikot Ide village, Nto Edino Clan,
in Obot Akara Local Government Area of Akwa Ibom
State, Nigeria. The leaves were thoroughly washed with
clean water and dried under shade for seven days to
reduce moisture content and prevent enzyme action.
The dry leaves were pulverized using mortar and pestle
into powdered form and then preserved in a black airtight polythene bag and stored away from light to
prevent it from photolysis until required.
A KeSoBAP Publication 2013. All rights reserved.

Evaluation of Prophylactic (Repository) Activities of

Extracts
The repository activity of the extract was assessed using
the method described by Peters (1965).
Thirty (30) mice were randomly divided into five
groups of six mice each. Group A mice received 10
ml/kg of distilled water (negative control). Mice in
groups B, C and D mice received 430, 860 and 1290
mg/kg body weight of the extract, respectively. Group E
mice received 1.2 mg/kg of Artesunate (positive
control). The treatment was carried out for three
consecutive days (Do D2) between 8 am and 9 am,
using oral route of administration.
On the fourth day (D3), the mice were inoculated with
Plasmodium berghei. The parasitaemia level was
assessed by blood smears seventy-two hours later.

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Evaluation of Suppressive Activity of the Methanol
Extracts (4-Day Test)
The schizontocidal activity of the extract, distilled water
and artesunate against early P. berghei infection in mice
was evaluated using the method described by Knight
and Peters (1980). Another thirty (30) mice were
randomly divided into five groups of six mice each. On
the first day, (Do), the mice were infected with the
parasite and randomly divided into various groups.
These animals were given the extracts, distilled water
and artesunate orally using oral carnula.

were fasted overnight and were grouped in threes. In

the initial phase, four groups of the mice were
administered with various doses of the crude methanol
extract at the dose of 1.0, 2, 3.0 and 4.0g/kg body
weight orally. The treated mice were monitored for
24hours for mortality and general behaviour. After
24hours, the remaining six groups of the mice were
treated with the dose of 4.5, 4.6, 4.7, 4.8, 4.9 and 5.0g/kg
body weight based on the findings of the phase 1 and
were monitored for another 24hours (Lorke, 1983 and
Bruce, 1985).
2.7 Ethical approval

Group A mice received 10 ml/kg of distilled water

(negative control); mice in groups B, C and D received
430, 860 and 1290 mg/kg body weight of the extract,
respectively. Group E mice received 1.2 mg/kg of
Artesunate (positive control). Dosing was done for four
consecutive days (Do D3) between 8 am and 9 am. On
the fifth day, thick blood films were made from tail
blood. The films were then stained with Leishmans
stain to reveal parasitized erythrocytes out of 500 in a
random field of the microscope. The average percentage
suppressive effect of parasitaemia was calculated in
comparison with the controls.
Evaluation of Curative Activities of Methanol
Extracts (Ranes test)
Ranes test was used to evaluate the schizonticidal
activity of the extract and artesunate in established
infection. This was done as described by Ryley and
Peters (1970). P. berghei berghei was injected
intraperitoneally into another 30 mice on the first day
(Do). Seventy-two hours later (D3), the mice were
divided randomly into five groups of six mice each and
administered orally.
Group A mice received 10ml/kg of distilled water
(negative control); mice in groups B, C and D received
430, 860 and 1290 mg/kg body weight of the extract,
respectively. Group E mice received 1.2mg/kg of
Artesunate (positive control). Dosing was done for four
consecutive days (Do D3) between 8am and 9am. On
the fifth day, thick blood film was made from tail blood.
The film was then stained with Leishmans stain to
reveal parasitized erythrocytes out of 500 in a random
field of the microscope. The average percentage
suppression of parasitaemia was calculated in
comparison with the control.
2.6 Acute toxicity studies
Experimental Animals
Albino mice (30) of either sex weighing 16-23 g were
obtained from the animal house of biological Science
Department, University of Calabar and were housed in
cages under standard condition (12 hours light and 12
hours dark), humidity and temperature in the
University of Uyo animal house, Department of
Pharmacology and Toxicology. They were fed on
growers palletized feed and water.
Lethal dose Determination (LD50)
The up and down method (Bruce, 1985) was used to
determine the toxicity level of the extract. The mice
A KeSoBAP Publication 2013. All rights reserved.

Permission and approval for animal studies were

obtained from the College of Health sciences animal
ethics committee, University of Uyo.
3. Results
1.0 kg of the powdered leaves of Nauclea latifolia
yielded 74 g (7.4%) of the methanol dried extract.
Phytochemical screening revealed that the methanol
extract of the leaf of Nauclea latifolia contained
alkaloids, cardiac glycosides, terpenes, polyphenols,
saponins, tannins and flavonoids in high concentration,
carbohydrates in moderate concentration and
anthraquinones in low concentration. Cyanogenic
glycosides, phlobatanins, balsams and resins were
absent (Table 1).
Table 1: Phytochemical analysis of the methanol
extract of Nauclea latifolia leaves
Constituent

Concentration in Leaf

Alkaloids

+++

Cardiac glycosisides

+++

Terpenes

+++

Cyanogenic glycosides
Carbohydrates
Anthraquinones

++
+

Polyphenols

+++

Saponins

+++

Tannins

+++

Flavonids

+++

Phlobatanins

Balsams

Resins

Key : +++ = present in high concentration

++ = present in moderate concentration
+ = present in low concentration
- = absent

The prophylactic / repository activity of methanol leaf

extract of Nauclea Latifolia showed a dosedependent
reduction in parasitaemia in the extract treated groups
(Table 2). These reductions were statistically
significant relative to control (p < 0.05) but not as much

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as that exhibited by the standard drug artesunate at 1.2
mg/kg.

0.05) but not as much as that of the standard drug artesunate

The chemosuppressive activity of methanol leaf extract

of Nauclea latrofolia on parasitaemia increased as the
dose of the extract increased (Table 3). These effects
were statistically significant relative to control (p <

The mean survival time of mice receiving methanol leaf

extract of Nauclea latifolia increased as the dose of the
extract increased. The increases were statistically
significant (p < 0.05) compared to control but not as
much as that of the standard drug, artesunate (Table 4).

Table 2: Prophylactic/repository activity of methanol leaf extract of Nauclea Latifolia on Plasmodium berghei berghei
infection in mice
Treatments

Group

Doses (mg/kg)

Parasitaemia

% chemosuppression

Distilled water
(Negative control)

10 ml/kg

17.430.34

Extract

430

10.520.24

40.1

860

8.240.28

53.0

1290

6.210.44

64.4

1.2

3.010.06

85.2

Artesunate

Values are expressed as mean SEM significance relative to control *p<0.05, (n=6)

Table 3: Suppressive activity of methanol leaf extract of Nauclea latrofolia on Plasmodium bergehei berghei infection
in mice 4 (day test)
Treatments
Distilled water
(Negative control)

Extract

Artesunate
(Positive control)

Group

Doses

Parasitameia

% Chemosuppression

10 ml/kg

22.021.05

430

11.98 0.35

46.6

860

10.500.46

56.0

1290

7.20 0.90

66.7

1.2

3.700.65

83.5

Values are expressed as mean SEM significance relative to control * P<0.05, (n=6)

Table 4: Mean survival time of mice receiving methanol leaf extract of Nauclea latifolia during established P. berghei
berghei infection in mice
Treatments

Doses (mg/kg)

Mean survival time (days)

Normal saline

10 ml/ kg

10.36 0.37

430

16.63 0.74

860

17.70 0.35

1290

22.33 0.86

1.2

26.28 0.51

Extract

Artesunate (positive control)

Values are expressed as mean SEM. Significance relative to control: P < 0.05

The maximum dose producing 0% mortality was 4.0

and the minimum dose producing 100% mortality was
4.7g/kg body weight. The LD50 was calculated to be 4.3
g/kg. The physical signs of toxicity observed included
excitation, paw licking, increased respiratory rate,
decreased motor activity, gasping, coma and death.

A KeSoBAP Publication 2013. All rights reserved.

4 . Discussion
In traditional medicine the leaf of Nauclea latifolia has
been used in the treatment of malaria, skin rashes,
jaundice, fever, oedema, haemolysis, dysentery, hernias,
diarrhoea and sexually transmitted diseases (Burkill,
1977). The folkloric use of this plant prompted the need

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Udobre et al. Afr. J. Pharmacol. Ther. 2013. 2(3): 83-87

to evaluate the in vivo antiplasmodial activity of its
methanol leaf extract to provide some support for its
ethnobotanical uses.

Burkill HM. (1997). Medicinal Importance of Nauclea latifolia.

The useful plants of West Tropical Africa. Vol. 4, Families M-R,
Kew: Royal Botanic Gardens. pp. 614-621.

The extract reduced the parasitaemia in suppressive,

prophylactic and curative models in a dose-dependent
manner. These results may support the reported
activity of the plant against norciception, inflammation,
pyrexia which are symptoms uncomplicated malaria
(Abbah et al, 2010)

Christensen SB and Kharazmi A (2001). Antimalarial Natural

Products. Isolation, Characterization and Biological
Properties. In: Bioactive Compounds from natural sources. .
Isolation, Characterization and Biological Properties. Tringali
C., editor, London: Taylor and Francis, pp.379-432
Joda AE, Kola-Mustapha AT, Folawewo I, Fetto BE and Quadric
UF. (2004). A review of malaria in Nigeria. Nigerian J. Pharm.
37: 45-54.

Some secondary metabolites of plants such as alkaloids

and terpenes have been associated with antiplasmodial
activity (Ponarulselvam et al, 2012, Christensen and
Kharazmi, 2001). In this study alkaloids and terpenes
were present in abundance. Monoterpene indolealkaloids have been implicated in endoperoxidation by
generating reactive oxygen species which in turn may
alkylate with heme protein resulting in the death of the
parasite (Robert, 1998; Shigemori et al, 2003).

Madubunyi I. (1995). Antihepatoxic and Trypanocidal

Activities of the Ethanolic Extract of Nauclea latifolia Root
Bark. J. Herbs, Species and Med. Plants. 3: 23-35.

5. Conclusion

Odetola A, Basir O. (1980). Evaluation of Antimalarial

properties of some Nigerian Medicinal Plants. In: Safowora, A.,
Editor, 275-283.

The Annang people in Akwa Ibom State, Nigeria drink

the leaf decoction of Nauclea latifolia for prevention and
cure of malaria. The result of this study has provided
some scientific basis for its ethnomedical use and also
supports the need for continued search for the active
principle responsible for the antimalarial property for
possible characterization.

Lorke D (1983). A new Approach to Practical Acute Toxicity

Testing. Arch. Toxicol. 53:275-287

Peters W. (1965). Drug resistance in Plasmodia berghi, Exp.

Parasitol. 17: 80-89.
Roberts F, Robert C and Johnson (1998). Evidence for
Shikimate Pathway of apicomplexan parasites. Nature, 393:
219-220
Ryley JF, Peter W. (1970). The antimalarial activity of some
quinolone esters. Annals Trop. Med. Parasitol. 84: 209-222.

Conflict of Interest declaration

The authors declare no conflict of interest

Sandberg, F and Bruhm, J.G. (1976). Screening of Plants for

Biologically Active Substances. African Med. Plants, Ife:
Obafemi Awolowo University Press, p. 119.

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A KeSoBAP Publication 2013. All rights reserved.

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